Genetically transformed rose plants and methods for their production

ABSTRACT

Rose plant cells are transformed by incubation with Agrobacterium cells carrying an exogenous DNA sequence. The callus cells may be obtained from various tissue sources, including stamen filaments, leaf explants, and the like, and whole rose plants may be regenerated from the transformed callus cells. The exogenous DNA will be stably incorporated into the chromosomes of the regenerated rose plant which will be able to express gene(s) encoded by the DNA sequence.

This application is a division of application Ser. No. 08/154,143, filedon Nov. 18, 1993, now U.S. Pat. No. 5,480,789; which in turn is acontinuation of application Ser. No. 07/678,846, filed Apr. 1, 1991, nowabandoned.

The subject matter of the present invention is related to that ofapplication Ser. No. 07/542,841, filed Jun. 25, 1990, the fulldisclosure of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to methods for geneticallyaltering the cells of higher plants. More particularly, the inventionrelates to a method for genetically transforming cells from rose plants.

The hybrid tea rose, Rosa hybrida, is one of the most popular of allcultivated plants. As with any valuable plant species, breeders havelong been working to improve existing varieties and create new varietiesusing conventional cross-breeding techniques. Characteristics ofparticular interest include color, fragrance, morphology, herbicideresistance, pesticide resistance, environmental tolerance, vase life ofthe cut flower, and the like. While improvements and variations in mostor all of these areas have been achieved, progress is slow because ofthe perennial nature of the plant and the high incidence of plantsterility caused by abnormal chromosome numbers. While rose tissueculture is now possible based on work described in co-pendingapplication Ser. No. 542,841, referenced above, the natural geneticvariation offered by tissue culture is random and still requiressubstantial effort to produce a particular genetic variation.

For these reasons, it would be desirable to use recombinant DNAtechnology to produce new rose cultivars in a controlled and predictablemanner. It would be particularly desirable to be able to geneticallytransform individual rose plant cells to introduce a desiredcharacteristic and to be able to regenerate viable somatic embryos androse plantlets from the modified cells. Such methods should be capableof introducing preselected exogenous genes to the rose plant cell andshould permit selection of transformed cells which are capable ofexpressing the gene. The method should produce regenerated rose plantswhich have stably incorporated the gene(s).

2. Description of the Background Art

Abstract A203 (Noriega et al.) in Abstracts VIIth International Congresson Plant Tissue and Cell Culture, Amsterdam, Jun. 24-29, 1990, reportspreliminary results on the production of calli from rose (Rosa hybrida)leaves. The reported results correspond to work described in relatedapplication U.S. Ser. No. 542,841, previously incorporated herein byreference.

Tissue culture methods involving Rosa hybrida and other rose species aredescribed in Handbook of Plant Cell Culture, Ammirato et al. (eds.),Chapter 29, 716-743, McGraw-Hill (1990); Skirvin et al. (1979) HortSci., 14:608-610; Hasegawa (1979) Hort Sci., 14:610-612; KhoshKhui etal. (1982) J. Hort Sci., 57:315-319; Valles (1987) Acta Horticulturae,212:691-696; Lloyd et al. (1988) Euphytica, 37:31-36; Burger (1990;)Plant Cell Tissue and Organ Culture, 21:147-152; Ishioka et al. (1990)Plant Cell, Tissue and Organ Culture, 22:197-199; Matthews et al. "AProtoplast to Plant System in Roses", 7th IAPTC Congress, Amsterdam; andde Wit et al. (1990) Plant Cell Reports, 9:456-458.

The susceptibility of certain Rosa species to infection and tumorinduction by Agrobacterium tumefaciens is described in De Cleene et al.(1976) The Botanical Review, 42:389-466. The susceptibility of certainRosa species to infection and hairy root induction by Agrobacteriumrhizogenes is described in De Cleene et al. (1981) The Botanical Review,47:147-194.

The transformation of embryogenic calli from Prunus persica (a member ofthe Rosaceae family) with Agrobacterium tumefaciens is reported inScorza (1990) In Vitro Cell Dev. Biol., 26:829-834. No disclosure oftransformed plant material beyond callus stage or of regeneration ofwhole plants is provided. The transformation of explant materials fromother members of the Rosaceae family is described in James et al. (1989)Plant Cell Reports, 7:658-661, and Graham et al. (1990) Plant Cell,Tissue and Organ Culture, 20:35-39.

The transformation of crushed tobacco callus with wild type (virulent)Agrobacterium tumefaciens resulting in crown gall formation is reportedin Muller et al. (1984) Biochem. and Biophys. Res. Comm., 123:458-462.

SUMMARY OF THE INVENTION

The present invention comprises methods for genetically transformingrose plant callus cells and, in the preferred embodiments, forregenerating the transformed callus cells into somatic embryos andultimately back into viable rose plantlets. The callus cells aretransformed by incubation with Agrobacterium cells carrying an exogenousDNA sequence which typically includes a selectable marker gene as wellas one or more genes to be expressed. Transformed callus cells areselected, typically on a medium which inhibits growth in the absence ofthe marker, and may be regenerated into somatic embryos and plantletswhich stably incorporate the DNA sequence(s).

The present invention further comprises rose callus cells, somatic roseembryos, and rose plantlets which incorporate exogenous DNA sequences.Preferably, such transformed cells, embryos, and plantlets are obtainedby the methods of the present invention.

The methods of the present invention provide a particularly convenienttechnique for selectively breeding new rose cultivars in a predictableand expeditious manner. It is expected that a variety of traits, such ascolor, fragrance, morphology, herbicide resistance, pesticideresistance, flower vase life, environmental tolerance, otherhorticultural traits, and may be intentionally introduced into thecallus cells and stably incorporated into the chromosomes of theregenerated embryos and plantlets.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a map of binary plasmid pJJ3499 used in Example 1 in theExperimental section hereinafter.

FIG.2 illustrates the T-DNA region of plasmid pJJ3491 used in Example 2of the Experimental section hereinafter. Plasmid pJJ3931 carries anos/NPT fusion and a 35S/luciferase fusion.

FIG. 3 is a bar graph of luminescence measurements from transformed roseembryogenic calli bearing the firefly luciferase gene. Fifteen putativetransformed calli (no. 1-15) and 12 non-transformed control calli(designated by C) were placed individually in 60 μl of 200 μM luciferinsolution in 1.5 ml microcentrifuge tubes for 30 minutes in the dark. Thetubes then were placed in scintillation vials and measured in ascintillation counter (Packard Instrument Co., Downers, Grove, Ill.,USA). The bars represent the number of light units emitted from eachsample in terms of log scale of cpm (counter per minute). The assay wasperformed generally as described in Ow et al. (1986) Science234:856-859.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

According to the present invention, genetically transformed rose plants,cells, and embryos are obtained by the selective introduction ofexogenous DNA sequence(s) into the chromosomes of cultured rose calluscells. The methods require certain starting materials, including asource of rose plant material to produce callus cells, the DNAsequence(s) to be introduced, Agrobacterium cells to carry the DNAsequence(s) and mediate their transfer to the rose callus cells, andculture media suitable for the steps of callus induction, DNA transfer,and embryo and plantlet regeneration, as described in much greaterdetail hereinbelow. Each of the necessary starting materials will now bedescribed.

The following terms, as used in the specification and claims, areintended to have the following meanings.

Somatic embryo: Structures similar to zygotic embryos which arise fromsomatic cells.

Embryonic: Capable of becoming somatic embryos.

In rose calli have surface structures (e.g., about 0.5 mm to 1 mm) whichare capable of becoming embryos.

Pre-embryogenic: Capable of becoming embryogenic. In rose, these calliare friable, whitish-creamish, granular.

Callus: Undifferentiated cell mass produced usually by culture ofdifferent organs in vitro. It can be hard, soft, dispersible, compact,spongy, dry, watery, or etc.

Callus Structures: See above.

Somatic Cell: Any of the body of an organism except the germ cells(sexual reproductive cells).

Rose plant tissue which is used for producing callus cells may beobtained from any species of the rose genus, Rosa. Exemplary speciesinclude Rosa damascena, Rosa multiflora, Rosa gallica, Rosa hybrida, andthe like. Of particular interest are various cultivars of Rosa hybrida,such as Royalty, Frisco, Sonia, and the like.

The plant tissue used for the production of callus cells may be matureor immature, preferably being mature somatic tissue. Suitable immatureplant tissue can be obtained from in vitro plant tissue culturetechniques, such as those described in Ammirato et al. (eds), Handbookof Plant Cell Culture, vol. 5, McGraw-Hill Publishing Co., New York,1990, particularly at Chapter 29, pages 716-747, the disclosure of whichis incorporated herein by reference. Callus cells obtained from tissueculture materials may be subjected to a "cell suspension" step prior totransformation as described below. Such cell suspension comprisessuspending the cells in a liquid culture medium and shaking thesuspension, typically at about 100 to 500 rpm. In some cases, cellsuspension may be useful to the production of embryonic cells.

The preferred mature somatic plant tissues may be obtained from any partof the mature rose plant that is capable of producing calli. Suitableplant parts include stamen filaments, leaf explants, stem sections,shoot tips, petal, sepal, petiole, peduncle, and the like, with stamenfilaments and leaf explants being particularly preferred.

Generally, the mature plant tissue sources will be disinfected prior tointroduction to the callus induction culture. A suitable disinfectionstep comprises an alcohol wash, e.g., with 75% ethanol for about oneminute, followed by a wash with bleach (10%) and a suitable detergent,e.g., 0.1% Tween®, for 20 minutes. The plant materials are then rinsed,usually two to three times for about five minutes each time, withsterile, deionized water prior to culturing.

Suitable stamen filaments will have a length from about 0.5 to 1.5 cm,preferably being about 1 cm. The stem and leaf sections are preferablycut to a size below about 1 cm×1 cm, preferably being about 0.5 cm×0.5cm. Shoot tips will be cut to a length in the range from about 0.5 to 3mm, preferably being about 1 mm in length.

The exogenous DNA sequences to be introduced will usually carry at leastone selectable marker gene to permit screening and selection oftransformed callus cells (i.e., those cells which have incorporated theexogenous DNA into their chromosomes), as well as one or more"functional" genes which are chosen to provide, enhance, suppress, orotherwise modify expression of a desired trait or phenotype in theresulting plant. Such traits include color, fragrance, herbicideresistance, pesticide resistance, disease resistance, environmentaltolerance, morphology, growth characteristics, and the like.

The functional gene to be introduced may be a structural gene whichencodes a polypeptide which imparts the desired phenotype.Alternatively, the functional gene may be a regulatory gene which mightplay a role in transcriptional and/or translational control to suppress,enhance, or otherwise modify the transcription and/or expression of anendogenous gene within the rose plant. It will be appreciated thatcontrol of gene expression can have a direct impact on the observableplant characteristics. Other functional "genes" include sense andanti-sense DNA sequences which may be prepared to suppress or otherwisemodify the expression of endogenous genes. The use of anti-sense isdescribed generally in van der Krol et al., (1990) Mol. Gen. Genet.220:204-212, the disclosure of which is incorporated herein byreference. The use of sense DNA sequences is described in variousreferences, including Napoli et al. (1990) Plant Cell, 2:279-289 and vander Krol et al. (1990) Plant Cell, 2:291-299, the disclosures of whichare incorporated herein by reference.

Structural and regulatory genes to be inserted may be obtained fromdepositories, such as the American Type Culture Collection, Rockville,Md. 20852, as well as by isolation from other organisms, typically bythe screening of genomic or cDNA libraries using conventionalhybridization techniques, such as those described in Maniatis et al.,Molecular Cloning--A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. (1985). Screening may be performed by (1)nucleic acid hybridization using homologous genes from other organisms,(2) probes synthetically produced to hybridize to particular sequencescoding for desired protein sequences, or (3) DNA sequencing andcomparison to known sequences. Sequences for specific genes may be foundin various computer databases, including GenBank, National Institutes ofHealth, as well as the database maintained by the United States PatentOffice.

The genes of interest may also be identified by antibody screening ofexpression libraries with antibodies made against homologous proteins toidentify genes encoding for homologous functions. Transposon tagging canalso be used to aid the isolation of a desired gene. Transposon taggingtypically involves mutation of the target gene. A mutant gene isisolated in which a transposon has inserted into the target gene andaltered the resulting phenotype. Using a probe for the transposon, themutated gene can be isolated. Then, using the DNA adjacent to thetransposon in the isolated, mutated gene as a probe, the normalwild-type allele of the target gene can be isolated. Such techniques aretaught, for example, in McLaughlin and Walbot (1987) Genetics,117:771-776; Dooner et al. (1985) Mol. Gen. Genetics, 200:240-246; andFederoff et al. (1984) Proc. Natl. Acad. Sci. USA, 81:3825-3829, thedisclosures of which are incorporated herein by reference.

Particular genes which may be incorporated into rose callus cellsaccording to the method of the present invention include the chalconesynthase gene (Napoli et al. (1990) Plant Cell 2 279:289) and the insectresistance gene (Vaeck et al. (1987) Nature 328:33).

The selectable marker gene on the DNA sequences to be inserted willusually encode a function which permits the survival of transformedcallus cells in a selective medium. Usually, the selectable marker genewill encode antibiotic resistance, particularly kanamycin resistance,hygromycin resistance, streptomycin resistance, chlorosulfuronresistance, (herbicide resistance), or the like. The composition of asuitable selective medium is described hereinbelow.

In addition to the "functional" gene and the selectable marker gene, theDNA sequences may also contain a reporter gene which facilitatesscreening of the transformed callus cells and plant material for thepresence and expression of the exogenous DNA sequences. Exemplaryreporter genes include β-glucuronidase and luciferase, as described inmore detail hereinafter.

The exogenous DNA sequences will be introduced to the callus cells byincubation with Agrobacterium cells which carry the sequences to betransferred within a transfer DNA (T-DNA) region found on a suitableplasmid, typically the Ti plasmid. Ti plasmids contain two regionsessential for the transformation of plant cells. One of these, the T-DNAregion, is transferred to the plant nuclei and induces tumor formation.The other, referred to as the virulence (vir) region, is essential forthe transfer of the T-DNA but is not itself transferred. By insertingthe DNA sequence to be transferred into the T-DNA region, introductionof the DNA sequences to the plant genome can be effected. Usually, theTi plasmid will be modified to delete or inactivate the tumor-causinggenes so that they are suitable for use as vector for the transfer ofthe gene constructs of the present invention. Other plasmids may beutilized in conjunction with Agrobacterium for transferring the DNAsequences of the present invention to callus cells.

The construction of recombinant Ti plasmids may be accomplished usingconventional recombinant DNA techniques, such as those described inManiatis et al., supra. Frequently, the plasmids will include additionalselective marker genes which permit manipulation and construction of theplasmid in suitable hosts, typically bacterial hosts other thanAgrobacterium, such as E. coli. Suitable selective marker genes includetetracycline resistance, kanamycin resistance, ampillcilin resistance,and the like.

The genes within the DNA sequences will typically be linked toappropriate transcriptional and translational control sequences whichare suitable for the rose plant host. For example, the gene willtypically be situated at a distance from a promoter corresponding to thedistance at which the promoter is normally effective in order to ensuretranscriptional activity. Usually, a polyadenylation site andtranscription termination sites will be provided at the 3'-end of thegene coding sequence. Frequently, the necessary control functions can beobtained together with the structural gene when it is isolated from atarget plant of other host. Such intact genes will usually includecoding sequences, intron(s), a promoter, enhancers, and all otherregulatory elements either upstream (5') or downstream (3') of thecoding sequences.

Optionally, a binary vector system may be used to introduce the DNAsequences according to the present invention. A first plasmid vectorstrain would carry the T-DNA sequence while a second plasmid vectorwould carry a virulence (vir) region. By incubating Agrobacterium cellscarrying both plasmids with the callus cells, infection of the calluscells can be achieved. See, Hoekema et al. (1983) Nature 303:179-180,the disclosure of which is incorporated herein by reference.

Suitable Agrobacterium strains include Agrobacterium tumefaciens andAgrobacterium rhizogenes. While the wild-type Agrobacterium rhizogenesmay be used, the Agrobacterium tumefaciens should be "disarmed," i.e.,have its tumor-inducing activity removed, prior to use. PreferredAgrobacterium tumefaciens strains include LBA4404, as described byHoekema et al. (1983) Nature, 303:179-180, and EHA101 (Hood et al.(1986) J. Bacteriol., 168:1291-1301. A preferred Agrobacteriumrhizogenes strain is 15834, as described by Birot et al. (1987) PlantPhysiol. Biochem., 25:323-325.

After the Agrobacterium strain(s) carrying the desired exogenous DNAsequence(s) have been prepared, they will usually be cultured for aperiod of time prior to incubation with the rose callus cells.Initially, the Agrobacterium may be cultured on a solid media includingnutrients, an energy source, and a gelling agent. Suitable nutrientsinclude salts, tryptone, and yeast extracts, while most sugars aresuitable as the energy source and the gelling agent can be agar,Gel-rite®, or the like. A preferred medium is L-Broth, which isdescribed in detail in the Experimental section hereinafter. Usually,medium will include an antibiotic to select for Agrobacterium carryingthe plasmid DNA sequences.

The Agrobacterium cells are typically cultured for about one to threedays, preferably in the dark at about 28° C., and are collected whilestill a white-creamish color, i.e., before browning, typically by beingscraped off the solid medium. The cells are then suspended in a liquidmedium, e.g., L-broth, or more preferably in an induction brothcontaining the following components:

    ______________________________________                                                    Broad Range  Preferred                                            ______________________________________                                        Ammonium chloride                                                                           0.5-3      g/l     1    g/l                                     Magnesium sulfate                                                                           0.5-3      g/l     1    g/l                                     Potassium chloride                                                                          0.05-2     g/l     0.15 g/l                                     Calcium       2-20       mg/l    10   mg/l                                    Ferrous sulfate                                                                             0.5-10     mg/l    2.5  mg/l                                    Phosphate monobasic                                                                         50-1000    mg/l    272  mg/l                                    MES           1000-10,000                                                                              mg/l    3904 mg/l                                    Glucose       2-30       g/l     5    g/l                                     Acetosyringone                                                                              10-200     μM   100  μM                                   Sucrose       10-30      g/l     20   g/l                                     pH            5-7                5.5                                          ______________________________________                                    

The Agrobacterium cells are cultured in the L-broth or induction brothfor about one to ten hours, preferably from about two to three hours,while being agitated, preferably at moderate temperatures from about 20°C. to 30° C.

Rose callus cells which may be transformed according to the method ofthe present invention may be produced as described in copendingapplication Ser. No. 542,841, the disclosure of which has previouslybeen incorporated herein by reference. Rose tissue is obtained from anyof the plant parts described above and placed in a callus inductionmedium including suitable nutrients, an energy source, growthregulators, and the like, selected to induce callus formation in theplant material. A variety of basal nutrient media are known whichprovide adequate supplies of nitrogen and salts to support callusgrowth, such as White's, B5, N6 and MS medium. Any sugar may be employedas energy source. Among the appropriate choices are glucose, maltose,sucrose, or lactose, or sucrose in combination with any of the namedsugars, or mannose. A preferred sugar for this purpose is sucrose, at alevel of about 10-50 g/l, but molar equivalents of other sugars may alsobe employed.

Callus induction medium preferably contains at least one auxin and atleast one cytokinin. The auxins may be any auxin, natural or synthetic,for example, indole acetic acid (IAA), naphthalene acetic acid (NAA),2,4dichlorophenoxy acetic acid (2,4-D), picloram, and dicamba. Thecytokinin may be selected from any of the known cytokinins, natural orsynthetic, for example 6-benzyladenine (6-BA), zeatin (ZEA), kinetin(KIN), and isopentyladenosine (iP). Callus may be induced in thepresence of several combinations of auxin and cytokinin. However,superior results are observed on an induction medium comprising 2,4-Dand zeatin. An alternate useful combination is NAA with kinetin.Generally, an auxin will be present in an amount of about 0.1 to 10mg/ml, and cytokinin in an amount of about 0.2 to 15.0 mg/ml. When theauxin is NAA, the concentration in the medium is preferably from about0.5 to 2.5 mg/ml, and most preferably about 2.0 mg/ml. When 2,4-D isused, the amount is preferably from about 0.5 to 10.0 mg/ml and mostpreferably about 2.5 mg/ml. When the cytokinin is kinetin, theconcentration in the medium is preferably from about 0.5 to 5 mg/ml andmost preferably about 0.5 mg/ml. When zeatin is used, the concentrationis preferably from about 0.2 to 12.5 mg/ml and most preferably about 1.5mg/ml. Other nonessential components may also be added to the medium tooptimize callus induction. For example, amino acids, such as glycine,may be employed as a nitrogen source. In certain embodiments, use ofadditional growth regulators may be helpful in promoting callusinduction. For example, addition of abscisic acid (ABA), in the amountof about 0.1 to 0.2 mg/l may be useful in callus induction, particularlyto promote a more globular callus, which leads to embryogenic tissue.ABA may be used with all explant sources, but has been especially usefulwith the culture of in vitro leaf explants.

Those skilled in the art will recognize that other components which arefrequently employed in plant tissue culture may also be incorporated inthe callus induction medium. Addition of various vitamins, e.g., MSvitamins, White vitamins, nicotinic acid, inositol, pyridoxine orthiamine is common. Similarly, for solid media, an appropriate amount ofsolidifying agent, such as agar or Gel-rite®, is also added to themixture.

The rose tissue is cultured on the callus induction medium for a timesufficient to produce at least one callus which serves as a source ofdispersed callus cells for transformation according to the presentinvention. Typically, tissue may be maintained in the callus inductionmedium for from about three to thirteen weeks, usually from about sevento ten weeks, and preferably for about eight weeks, to yield a fastgrowing callus. Initially, callus morphology may be hard, spongy,watery, sandy, or globular, and may have a white, cream, or yellowcolor, depending on the particular composition of the medium. Thepreferred morphology for use in the transformation methods of thepresent invention occurs after from about seven to ten weeks, usually atabout eight weeks, when the calli become highly friable or dispersablewith a whitish-creamish color and a granular consistency. While cellsfrom calli having these characteristics have been found to be mostsuitable, cells from calli which are hard and compact may also be usedfor transformation by cutting into small sections, typically havingdimensions of about 2 to 3 mm.

Calli cultured as just described may be used directly as the source ofcallus cells for transformation or may be subcultured prior to use as astarting material. Subculturing allows the continuing maintenance ofcallus cells as a source of starting materials for the method of thepresent invention.

In order to achieve the desired transformation, the callus materialdescribed above is incubated with the Agrobacterium cells carrying theexogenous DNA sequence to be transferred, typically for about one tofour days. Incubation is achieved in a cocultivation medium whichincludes nutrients, an energy source, and an induction compound which isselected to induce the virulence (vir) region of Agrobacterium toenhance transformation efficiency. The induction compound can be anyphenolic compound which is known to induce such virulence, preferablybeing acetosyringone (AS) present at from about 10 to 200 μM, preferablyat about 100 μM. Suitable phenolic compounds are described in Bolton etal. (1986) Science 232:983-985.

The preferred cocultivation medium includes sucrose (20 g/l) as theenergy source, 2,4-D (5 mg/l) as the auxin, and zeatin (1 mg/l) as thecytokinin. Gibberellic acid (1 mg/l) is also preferably present as agrowth regulator. A preferred formulation for the cocultivation mediumis N12AS set forth in the Experimental section hereinafter.

Callus cells are combined with the Agrobacterium cells in thecocultivation medium at a moderate temperature, typically in the rangefrom about 20° to 28° C., preferably at about 24° C., from about one tofour days, usually from about one to two days. The medium is preferablykept in the dark, and the cocultivation continued until theAgrobacterium have grown sufficiently so that colonies are observable onthe calli, either directly or through a microscope.

The Agrobacterium cells are present at a concentration from about 10⁷ to10¹⁰ cells/ml, preferably at about 10⁹ cells/ml. The callus cells arepresent at a ratio of from about 1:1 to about 10:1 (calluscells:Agrobacterium cells), preferably at about 3:1, on a volume basis.Usually, a total of about 1 to 100 ml of callus material is used,preferably about 10 ml, in a total culture volume of about 1 to 100 ml,preferably about 10 to 12 ml. Preferably, the callus cells andAgrobacterium cells are placed on a filter paper matrix, such as Whatman#1, on the cocultivation medium.

After transformation is completed, the callus cells are washed from theAgrobacterium cells with water or a culture medium containing nutrients,an energy source, growth regulators, and the like. For smaller callusstructures, typically in the range from about 0.2 to 0.3 mm in size, useof N12 medium (see the Experimental section hereinafter) is particularlysuitable. For larger callus structures, typically from about 0.4 to 0.7mm in size, use of M53 medium is particularly suitable.

The transformed calli are mixed with the wash medium, typically at avolume ratio of from about 1:3 to about 1:30 (calli:liquid), preferablyat about 1:10, and centrifuged, preferably at 500 rpm for about 5minutes. The resulting liquid fraction containing most of the bacteriais removed, while the denser fraction containing the calli is saved. Thewash is repeated, typically from two to six times, with antibioticsbeing used in at least the later washes in order to kill any remainingAgrobacterium cells. Any antibiotic capable of killing Agrobacterium maybe used, with carbenicillin (200 to 1000 mg/l), vancomycin (100 to 500mg/l), cloxacillin (200 to 1000 mg/l) cefotaxin (200 to 1000 mg/l), anderythromycin (200 to 1000 mg/l), being preferred.

After washing, the calli are placed on a suitable selection mediumincluding a plant selection agent which permits identification oftransformed calli based on the presence of the marker introduced as partof the exogenous DNA. Conveniently, the selective media is placed in apetri dish with portions of the calli, typically about 100 mg each. Theselection medium is a general growth medium, such as N12 or M53 (asdescribed in the Experimental section hereinafter) supplemented with theplant selection agent, and usually including the anti-Agrobacteriumantibiotic. Suitable plant selection agents include the following.

    ______________________________________                                                       Concentration of                                               Antibiotic Resistance                                                                          Antibiotic Selection Medium                                  ______________________________________                                        kanamycin        200-500     mg/l                                             hygromycin       20-80       mg/l                                             spectinomycin    20-80       mg/l                                             streptomycin     100-500     mg/l                                             chlorsulfuron    0.001-0.05  mg/l                                             ______________________________________                                    

Preferred selection media are N12 and M53 (see Experimental sectionhereinafter) containing no cytokinin or auxins, but having abscisic acidadded at from about 0.5 to 4 mg/l, preferably at about 2 mg/l. M53 (seeExperimental section hereinafter) is particularly preferred when thecallus structures are sized from about 0.4 to 0.7 mm. When kanamycinresistance is the selectable marker, N12CK and M53CK (see Experimentalsection hereinafter) are particularly suitable.

The selection culture will be maintained for a time sufficient to permittransformed callus cells to grow and produce white-cream colored calli,while the non-transformed callus cells turn brown and die. Typically,the selection culture will last from about 25 to 50 days, dependingprimarily on the concentration of the plant selective agent. Forexample, thirty days is generally sufficient for kanamycin at 300 mg/l,while fifty days is suitable for kanamycin at 200 mg/l. The primarycriterion in ending the selection culture, however, is a cleardistinction between proliferating cells which have been transformed andnon-proliferating cells which have not been transformed.

While viability is indicative that the callus cells have beentransformed, it is usually desirable to confirm transformation using astandard assay procedure, such as Southern blotting, Northern blotting,restriction enzyme digestion, polymerase chain reaction (PCR) assays, orthrough the use of reporter genes. Suitable reporter genes and assaysinclude β-glucuronidase (GUS) assays as described by Jefferson, GUS GeneFusion Systems User's Manual, Cambridge, England (1987) and luciferaseassays as described by Ow (1986) Science 234:856-859. It will beappreciated that these assays can be performed immediately following thetransformation procedures or at any subsequent point during theregeneration of the transformed plant materials according to the presentinvention.

Following transformation, the calli are transferred to a maintenancemedium for generation of somatic embryos. This medium contains as itsprinciple elements an auxin, a cytokinin, an energy source, and anappropriate nutrient medium such as White's or B5 media. The maintenancemedium will also include an anti-Agrobacterium antibiotic and, usually,ABA or gibberellic acid.

The formulation of the maintenance medium may be adjusted depending onthe source of somatic tissue. If the mature somatic tissue was obtainedfrom a stamen filament or cell suspension culture, the ratio of auxin tocytokinin may be decreased by a factor of at least two and up to as muchas 15 relative to the ratio of auxin to cytokinin present in callusinduction medium and/or the source of the auxin and cytokinin in theregeneration will differ from the source of the auxin and cytokinin inthe callus induction medium. In a preferred embodiment, a weakercytokinin and auxin is used in the regeneration media than in theinduction media and selection medium. Specifically, 2,4-D is a strongerauxin, i.e., has a greater effect on growth regulation than NAA andzeatin is a stronger cytokinin than kinetin. As an example, regenerationof filaments can occur in a medium comprising 2,4-D/zeatin at a ratio of1.3, compared with NAA/kinetin at a ratio of 4.0 in callus inductionmedium.

If the mature somatic tissue was obtained from a leaf explant, the ratioof auxin to cytokinin may be increased relative to the ratio of auxin tocytokinin present in callus induction medium and/or the source of theauxin and cytokinin in the regeneration medium will differ from thesource of the auxin and cytokinin in the callus induction medium. As anexample, regeneration of leaf explants can occur in a maintenance mediumcomprising NAA/KIN at a ratio of 2.0 compared with 2,4-D/zeatin at aratio of 1.3.

Preferred maintenance media are M53C (particularly if N12CK was theselection medium) and M20C (particularly if M53CK was the selectionmedium).

The period on maintenance medium for regeneration generally takes about20 to 60 days, usually about 30 days. Globular to heart-shaped embryoswill usually be apparent on the surface of the culture after this time.In many cases, the embryos so formed are capable, upon subculture, togive rise on their outer surface to secondary embryos. If this secondaryembryo production is specifically desired, the globular embryos can betransferred to fresh regeneration media and cultured from 3 to 6 weeks.

The somatic embryos produced on the maintenance medium as just describedcan be repeatedly subcultured in order to provide for an increasednumber of transformed embryos. In order to reproduce whole plantmaterial, however, it is desirable that the somatic embryos be subjectedto a maturation process.

Maturation of somatic embryos is accomplished by transfer of globularembryos to a medium comprising nutrients, an energy source, and a growthregulator which may include but is not limited to an auxin, a cytokinin,abscisic acid, and gibberellic acid. The auxins may be any auxin,natural or synthetic, for example, IAA, NAA, 2,4-D, and picloram. Theauxin will be present in an amount of about 0.1 to 10 mg/ml. Thecytokinin may be selected from any of the known cytokinins, natural orsynthetic, for example, 6-BA, ZEA, KIN, and iP. A cytokinin may bepresent in an amount of about 0.2 to 15.0 mg/ml. Abscisic acid may bepresent in the amount of about 0.2 to 2 mg/l. Gibberellic acid may bepresent in the amount of about 0.5 to 5 mg/l. A preferred maturationmedium is M20 (see Experimental section hereinafter).

Callus cells are held on the maturation medium with subculturingpreferably about every 30 days, until mature somatic embryos areobtained. The period of maturation generally takes about three to sixweeks. Globular embryos will appear on the surface of the maturationmedium, with many embryos giving rise on their outer surface tosecondary embryos. If such secondary embryo production is desired, theglobular embryos can be transferred to fresh maintenance medium (asdescribed above) and can be subcultured repeatedly in order to provide agreater number of embryos. Such subculturing is preferably performed onM20 medium.

The mature somatic embryos produced as described above are nexttransferred to a germination medium in order to produce germinatedembryos. The germination medium comprises nutrients and an energysource. The medium may further comprise a growth regulator which mayinclude but is not limited to a cytokinin, abscisic acid, andgibberellic acid. The cytokinin may be present at a concentration ofabout 0.1 to 1.0 mg/l. Abscisic acid may be present in the amount ofabout 0.2 to 2 mg/l. Gibberellic acid may be present in the amount ofabout 0.5 to 5 mg/l. The germination media may also further comprisecoconut water at about 5 to 15%, v/v. A preferred germination medium isM13. The somatic embryos are held on the germination medium for fromabout 1 to 45 days, usually about 24 days, to yield germinated embryos.

Early stages of embryo germination are characterized by hypocotylelongation, cotyledonary leaves and chlorophyll development. In latestages of germination, cotyledonary leaves enlarge, the hypocotylelongates, and a tap root develops. The differentiated embryos may becultured on germination media for about 1 to 4 weeks. The result issomatic embryos with shoots 1 to 4 mm in length having from 2 to 4leaves.

Optionally, the germinated embryos may be transferred to a shootelongation medium to produce elongated shoots. The medium will includenutrients, an energy source, and growth regulators, generally asdescribed above, but will have a reduced salt concentration (up to 50%lower) and a reduced growth regulator content, preferably BA at 1 to 6mg/l and IAA at 0.1 to 1 mg/l. A preferred shoot elongation medium isM13-8 (see Experimental section hereinafter). The embryos are maintainedin the elongation medium until the shoots are about 10 to 20 mm inlength and develop three to five fully green and elongated leaves andstems, typically requiring three to four weeks.

The germinated (and optionally shoot elongated) embryos are subsequentlytransferred to a propagation (or shoot multiplication) medium whichcomprises appropriate nutrients, an energy source, an auxin, and acytokinin. The auxin may be any auxin, natural or synthetic, forexample, IAA, NAA, 2,4-D and picloram. The auxin will be present in anamount of about 0.1 to 10 mg/l. The cytokinin may be selected from anyof the known cytokinins, natural or synthetic, for example, 6-BA, ZEA,KIN, and iP. A cytokinin may be present in an amount of about 0.2 to15.0 mg/l. In a preferred embodiment, the auxin is IAA, present at aconcentration of about 0.3 mg/l and the cytokinin is 6-BA, present at aconcentration of about 3.0 mg/l. A preferred propagation or shootmultiplication medium is M13 (see Experimental section hereinafter).

The germinated embryo may be cultured in propagation medium for about 20to 200 days, preferably about 30 days. Well developed plantlets may beobtained and can be transferred to, for example, artificial soil forroot regeneration. In one embodiment, multiple shoots can be isolatedfrom one single plantlet before transferring to soil.

Well developed shoots, typically having a length in the range from about10 to 40 mm and preferably having from about 5 to 10 leaves, areselected for root regeneration. The preferred method for rootregeneration is to transfer the shoots to be rooted into small potscontaining an artificial soil, typically saturated with a mediumcontaining root inducing hormones. A suitable root induction containsnutrients but is deprived of sugar and other energy sources. The mediummay further contain thiamine, preferably in the form of thiamine-HCl atabout 0.5 to 2 mg/l, and an auxin, such as IAA at about 1 to 4 mg/l. Apreferred root regeneration medium N3-4 (see Experimental sectionhereinafter). While in the pots, the shoots may be placed in acontainer, such as a magenta GA-7 culture container and incubated in agrowth chamber preferably under a regime of 16 hours light per 24 hourperiod.

An alternate regeneration method is to dip the shoots in a suitableroot-inducing hormone, such as RooTone™. The shoots are then placeddirectly in the soil in the greenhouse, preferably being maintainedunder a plastic cover to maintain a high relative humidity. The covercan be gradually removed over a period of days in order to causehardening of the shoots.

With either of the above approaches, roots are typically obtained inabout 7 to 35 days. The rooted shoots can then be transplanted withinthe greenhouse or elsewhere in a conventional manner for tissue cultureplantlets.

Transformation of the resulting plantlets can be confirmed by assayingthe plant material for any of the phenotypes which have been introducedby the exogenous DNA. In particular, suitable assays exist fordetermining the presence of certain reporter genes, such asβ-glucuronidase and/or luciferase, as described hereinabove. Otherprocedures, such as PCR, restriction enzyme digestion, Southern blothybridization, and Northern blot hybridization may also be used.

The following examples are offered by way of illustration, not by way oflimitation.

    ______________________________________                                        EXPERIMENTAL                                                                  MATERIALS                                                                     Abbreviations/Names                                                                           Source/Reference                                              ______________________________________                                        ABA; Abscisic Acid                                                                            Sigma Chemical Co., St Louis, MO,                                             USA                                                           Acetosyringone  Aldrich Chemical Co., Milwaukee,                                              WI, USA                                                       Agar; TC Agar   Hazleton Biologics, Inc., Lenexa,                                             KS, USA                                                       As; Acetosyringone                                                                            Aldrich Chemical Co., Milwaukee,                                              WI, USA                                                       B-5 Salts       Gamborg et al. (1968) Exp. Cell                                               Res. 50:151-158                                               BA; Benzyl Adenine                                                                            Sigma Chemical co., St. Louis, MO,                                            USA                                                           Bactogar        Difco, Detroit, MI, USA                                       Carbenicillin   Geopen                                                        2,4-D; 2,4-Dichloro-                                                                          Sigma Chemical Co., St. Louis,                                phenoxyacetic Acid                                                                            MO, USA                                                       Dropp, a cotton Nor-Am Chemical Co., Wilmington,                              defoliant whose DE, USA                                                       active ingredient                                                             is thidauzuron                                                                GA.sub.3 ; Gibberellic Acid                                                                   Sigma Chemical Co., St. Louis, MO,                                            USA                                                           G418; Geneticin Sigma Chemical Co., St. Louis, MO,                                            USA                                                           Gel-rite ®  Scott Lab. Inc., Warwick, RI, USA                             GUS; β-glucuronidase                                                     IAA; Indole-3-Acetic                                                                          Sigma Chemical Co., St. Louis,                                Acid            MO, USA                                                       IBA; Indole Butyric                                                                           Sigma Chemical Co., St. Louis,                                Acid            MO, USA                                                       Insolitol       Sigma Chemical Co., St. Louis,                                                MO, USA                                                       Jiffy Mix       Ball Jiffy, Chicago, IL, USA                                  Jiffy Pots      Ball Jiffy, Chicago, IL, USA                                  Kanamycin, Kanamycin                                                                          Sigma Chemical Co., St. Louis,                                Sulfate         MO, USA                                                       KIN, Kinetin    Sigma Chemical Co., St. Louis,                                                MO, USA                                                       LUC, Luciferase Analytical Luminescence Lab, San                                              Diego, CA, USA                                                Luciferin, D-Luciferin-                                                                       Analytical Luminescence Lab,                                  sodium          San Diego, CA, USA                                            MES, 2-N Morpholino-                                                                          Sigma Chemical Co., St. Louis,                                ethanesulfonic Acid                                                                           MO, USA                                                       MS Salts        JRH Bioscience, Lenexa, KS, USA                               MS Vitamins     Murashige, et al., Physiol. Plant                                             (1962) 15:473-497                                             N.sub.6  Salts  Chu, et al., Scientia Sinica                                                  (1975) 18:659-668                                             NAA, Naphthalene Acetic                                                                       Sigma Chemical Co., St. Louis,                                Acid            MO, USA                                                       Nicotinic Acid  Sigma Chemical Co., St. Louis,                                                MO, USA                                                       NPT, Neomycinphospho-                                                         transferase                                                                   Pyridoxine      Sigma Chemical Co., St. Louis,                                                MO, USA                                                       RooTone ™    Cooke Lab Products, Portland, OR,                                             USA                                                           TDZ, Thidiazuron                                                                              Purified from Dropp by dissolving                                             in dimethylsulfoxide and passing                                              through a 0.2 μm nylon filter.                             Tetracycline    Sigma Chemical Co., St. Louis, MO,                                            USA                                                           Thiamine-HCl    Sigma Chemical Co., St. Louis, MO,                                            USA                                                           Triton, TritonX-100                                                                           Sigma Chemical Co., St. Louis,                                                MO, USA                                                       Tryptone        Difco-Lab, Detroit, MI, USA                                   Tween ®     ICI United States, Inc.,                                                      Wilmington, DE, USA                                           Vancomycin      Sigma Chemical Co., St. Louis, MO,                                            USA                                                           Vitamins        Sigma Chemical Co., St. Louis, MO,                                            USA                                                           X-GUS, 5-Bromo-4-chloro-                                                                      Diagnostic Chem. Ltd., Monroe,                                3-Indolyl-β-D-                                                                           CT, USA                                                       Glucuronide                                                                   Yeast Extract   Difco-Lab, Detroit, MI, USA                                   Zeatin          Sigma Chemical Co., St. Louis, MO,                                            USA                                                           ______________________________________                                    

    ______________________________________                                        MEDIA COMPOSITIONS                                                            ______________________________________                                        M13                                                                           MS Salts               1x                                                     Thiamine HCl           0.5    mg/l                                            Inositol               100.0  mg/l                                            Pyridoxine             0.5    mg/l                                            Nicotine Acid          0.5    mg/l                                            Glycine                2.0    mg/l                                            BA                     3.0    mg/l                                            IAA                    0.3    mg/l                                            Aqar                   6.0    g/l                                             Sucrose                30     g/l                                             pH                     5.8                                                    M13-8                                                                         Same except:                                                                  MS Salts               3/4x                                                   Pyridoxine             1.5    mg/l                                            Nicotinic Acid         1.5    mg/l                                            M20 (alternatively M134-20)                                                   MS Salts               1x                                                     Thiamine HCl           5      mg/l                                            Inositol               100.0  mg/l                                            Pyridoxine             1.5    mg/l                                            Nicotinic Acid         1.5    mg/l                                            Glycine                2.0    mg/l                                            GA.sub.3               1.0    mg/l                                            ABA                    0.2    mg/l                                            KAO Vitamins*          1x                                                     Coconut Water**        10%    v/v                                             Sucrose                20     g/l                                             Gel-rite ®         2.4    g/l                                             pH                     5.5                                                    *Kao et al., 1975, Planta 126:105                                             **Not essential                                                               M20C                                                                          M20 plus carbenicillin 500    mg/l                                            M20K200C                                                                      M20C plus kanamycin    200    mg/l                                            M53                                                                           MS Salts               1x                                                     Thiamine HCl           5      mg/l                                            Inositol               20.1   g/l                                             Pyridoxine             1.5    mg/l                                            Nicotinic Acid         1.5    mg/l                                            Glycine                2.0    mg/l                                            GA.sub.3               1.0    mg/l                                            ABA                    2.0    mg/l                                            Sucrose                30     g/l                                             Gel-rite ®         2.4    g/l1                                            pH                     5.5                                                    M53AS                                                                         M53 plus As            100    μM                                           M53C                                                                          M53 plus carbenicillin 500    mg/l                                            M53CK                                                                         M53C plus kanamycin    300    mg/l                                            M130-3                                                                        MS salts               1x                                                     MS vitamins            1x                                                     Glycine                2      mg/l                                            KIN                    0.5    mg/l                                            NAA                    2      mg/l                                            Sucrose                30     g/l                                             Gel-rite ®         2.4    g/l                                             pH                     5.7                                                    M134-1                                                                        MS Salts               1x                                                     Thiamine-HCl           5      mg/l                                            Inositol               100    mg/l                                            Pyridoxine             1.5    mg/l                                            Nicotinic Acid         1.5    mg/l                                            Glycine                2      mg/l                                            Zeatin                 1.5    mg/l                                            NAA                    0.025  mg/l                                            GA.sub.3               1      mg/l                                            Sucrose                20     g/l                                             Gel-rite ®         2.4    g/l                                             pH                     5.7                                                    M139                                                                          B-5 salts              1x                                                     Ammonia Sulfate        329    mg/l                                            Thiamine-HCl           5      g/l                                             Inositol               100    mg/l                                            Pyridoxine             1.5    mg/l                                            Nicotinic Acid         1.5    mg/l                                            Glycine                2      mg/l                                            2,4-D                  1.55   mg/l                                            Sucrose                30     g/l                                             Gel-rite ®         2.4    g/l                                             pH                     5.6                                                    M139-2                                                                        M139 modified as follows:                                                     2,4-D                  2.0    mg/l                                            Zeatin                 1.5    mg/l                                            N3-1                                                                          N.sub.6  salts         1/2x                                                   Thiamine HCl           1.0    mg/l                                            Sucrose                20     g/l                                             Gel-rite ®         2.2    g/l                                             pH                     5.6                                                    N3-4                                                                          N3-1 modified as follows:                                                     NAA without sucrose    2.0    mg/l                                            and Gel-rite ®                                                            N12                                                                           N.sub.6  salts         1x                                                     Thiamine HCl           5      mg/l                                            Inositol               100.0  mg/l                                            Pyridoxine             1.5    mg/l                                            Nicotinic Acid         1.5    mg/l                                            Glycine                2.0    mg/l                                            2,4-D:                 5.0    mg/l                                            Zeatin                 1.0    mg/l                                            GA.sub.3               1.0    g/l                                             KAO Vitamins           1x                                                     Sucrose                20     g/l                                             Gel-rite ®         2.4    g/l                                             pH                     5.5                                                    N12AS                                                                         N12 plus As            100    μM                                           N12C                                                                          N12 plus cabenicillin  500    mg/l                                            N12CK                                                                         N12C plus kanamycin    300    mg/l                                            MinA                                                                          KH.sub.2 PO.sub.4      10.5   g/l                                             (NH.sub.4).sub.2 SO.sub.4                                                                            1.0    g/l                                             Sodium citrate.2H.sub.2 O                                                                            0.5    g/l                                             Agar                   15     g/l                                             L-Broth*                                                                      Tryptone               10     g/l                                             Yeast Extract          5      g/l                                             NaCl                   5      g/l                                             Glucose                1      g/l                                             Agar                   15     g/l                                             ______________________________________                                         *pH adjusted to 7.0 to 7.2 using 0.1-5N NaOH, before adding agar; dispens     at 25 ml/plate.                                                          

METHODS AND RESULTS Example 1 Agrobacterium rhizogenes Transformation ofRose

1. Culture tissue on callus induction medium to yield calli.

Stamen filaments of Rosa hybrida L. var. Royalty (obtained from DeVoreNurseries, Watsonville, Calif.) were excised from flower buds of ca. 1.5cm long, after a cold pretreatment at 2° C. during 14 days. Buds weredisinfected with clorox (10%)/Tween-®20 (0.1^(%)) for 20 mins., rinsedthree times with sterile deionized water and placed in callus inductionmedium (M130-3). All media were autoclaved for 20 min. at 24° C. and 15psi after pH adjustment. Cultures in petri dishes were sealed withParafilm and kept in the dark at 24° C. A fast-growing, semi-hard,yellow callus was obtained from filament explants after 3 weeks inM130-3. After subculture in this medium, the callus changed to a drierappearance.

The callus was placed in maintenance medium M139. M139 medium improvedcallus quality preventing oxidation and leading to a less compactcallus.

2. Pre-embryogenic callus induction medium and their maintenance.

M139 medium with modified growth regulators 2,4-D (2.0 mg/l) and zeatin(1.5 mg/l), was used as pre-embryogenic friable callus induction medium(M139-2). Early stages of pre-embryogenic calli were observed after 8weeks of callus culture on M139-2 at a frequency of 1.43%. Globularstructures were subcultured on a proliferation medium, M134. KM-8Pvitamins (Kao and Michayluk (1975) Planta, 126:105-110) and growthregulators were filter sterilized and added into the autoclaved portionof proliferation medium. After 3 weeks, a very fast-growing friable, andwhite embryonic tissue with the presence of globular structures wasproduced. Periodic subculture of this tissue on medium maintained itscapacity to proliferate and to produce globular structures. Such tissuewas able to be maintained on N12 medium for 8 months.

3. Agrobacterium rhizogenes culture and preparation.

Agrobacterium rhizogenes wild-type strain 15834 (Birot et al. (1987)Plant Physiol. Biochem. 25:323-325) containing the binary vector pJJ3499was used for transformation. pJJ3499 contains the nopaline synthasepromoter and neomycin phosphotransferase II (NPT II) gene which conferskanamycin resistance as well as the cauliflower mosaic virus 35Spromoter. The β-glucuronidase gene (Jefferson (1986) Proc. Natl. Acad.Sci. USA 83:8447-8451) is present as a reporter gene. Strain 15834 alonewas used as a control inoculum. Bacteria were maintained on L-brothmedium solidified with 1.5% Bactoagar containing 10 mg/l tetracycline.Bacteria were scraped off the solid medium using a loop and suspendedin"Induction Broth" medium (Winans et al. (1989) J. Bact. 171:1616-1622)containing 100 μM acetosyringone, and cultured on a shaker (120 rpm) at28° C. for 3 hours.

4. Cocultivation on cocultivation medium.

Agrobacterium cells were mixed at the volume ratio of 3:1 (plantcell:Agrobacterium cell) with the friable calli selected after 6 months.Calli and Agrobacterium were placed on 7.0 cm sterile Whatman #1 filterpaper circles on the top of cocultivation medium N12 supplemented with100 μM acetosyringone. Plates were placed in a 24° C. controlledenvironment incubator in the dark for 48 hours.

5. Wash.

Calli were washed from Agrobacterium with the liquid medium N12supplemented with 500 mg/l carbenicillin. Calli were mixed well with themedium at a volume ratio of 1:10 (calli:medium), centrifuged (500 rpmfor 5 min.), and the supernatant was discarded. Washing was repeated 4times.

6. Selection medium.

After washing, 10-12 chunks (about 100 mg each) of calli were placed andspread on selection medium N12CK containing 300 mg/l kanamycin sulfatefor selection and 500 mg/l carbenicillin to kill off the residualAgrobacterium. Tissues remained on this medium for 30 days. At the endof the 30 day culture period, most parts of the calli turned brown,however one to a few sections of each callus started growing to producewhite-cream colored calli. 75 out of 81 inoculated calli producedkanamycin-resistant calli (Table 1).

                  TABLE 1                                                         ______________________________________                                        Recovery of Kanamycin-Resistant Calli on N12 Medium                                                Number of                                                                     Chunks Plated                                                                 on Selection                                                                              Number of                                              Kanamycin  Medium After                                                                              Calli Growing                                Treatment Level (mg/l)                                                                             Cocultivation                                                                             1 month later                                ______________________________________                                        Inoculated                                                                              300        81          75                                           with 15834                                                                               0         23          23                                           (Example 1)                                                                   Inoculated                                                                              300        33          25                                           with LBA 4404                                                                            0         12          12                                           (Example 2)                                                                   Uninoculated                                                                            300        25           0                                           Control    0         15          15                                           ______________________________________                                    

7. Culture on maintenance medium to yield somatic embryos.

White-cream colored callus tissues were then transferred to N12C mediumcontaining 500 mg/l carbenicillin (but no kanamycin) or M53C for 23days. The tissue on N12C was then transferred to medium M53 for threeweeks. Calli proliferated further on these media and produced largerglobular structures.

8. Culture on maturation medium to yield somatic embryos.

The callus tissue from part 7 was subsequently cultured in maturationmedium M20 for either 8 or 11 weeks. On this medium, mature embryos wereobtained starting after four weeks and continuing afterwards. Matureembryos appeared on structures with wide cotyledons (usually 2 andoccasional 3 or 4) and very short hypocotyl and radical. The embryoswere white. The same results were obtained for both the 8 week and 11week culture period.

9. Culture on germination medium.

Germination of the matured embryonic tissue was accomplished on M13medium after 2 weeks. Under 16 hr/day light illumination (around 1500lux) tissues became green, cotyledons expanded 5-10 times, and embryosenlarged in size 3-5 times and produced 1-5 green shoots.

10. Culture on shoot multiplication medium.

Germination embryos were subcultured on fresh M13 medium. On this mediumshoots multiplied further, and after 4 weeks, ten to 30 shoots peroriginal embryo were produced.

11. Culture on shoot elongation medium.

Sections of the shoot clusters were cut off and transferred to M13-8medium with 4-6 shoots per cluster. Shoots elongated to 10-15 cm in sizewithin 3-4 weeks.

12. Culture on artificial soil for root regeneration.

Shoots were cultivated in Jiffy Mix saturated with N3-4 medium. After 6weeks, well developed shoots were obtained and were in condition fortransfer to artificial soil.

13. Culturing shoots in soil for root regeneration.

Shoots were dipped in RooTone™ and planted in a mix soil (3:1 SuperSoil: Perlett, Rod Mclellan Co., So. San Francisco, Calif. USA) ingreenhouse and watered as needed. After 3 weeks roots were regeneratedand complete transgenic plants were obtained. Plants were covered with aplastic sheet which was gradually (within 2 weeks) removed to harden offthe plants.

14. Results and demonstration of transformation.

Transformation was confirmed by several means: 1) transformed callitransferred onto M20K200C were able to continue their growth, whereasnontransformed control calli stopped growth on the medium, turned brownand eventually died (Table 2); 2) transformed calli, somatic embryos,and leaf sections from transformed shoots all tested positive andnontransformed controls tested negative in the GUS assays (Table 3)(transformants stained blue and nontransformed tissues did not stainblue).

Leaf callus assays were performed on five transgenic shoots. The mediumcontained 50 mg/l kanamycin to verify that the tissues had beentransformed. All transformants formed calli in the presence of thekanamycin, thus confirming transformation.

                  TABLE 2                                                         ______________________________________                                        Assay for Kanamycin Resistance                                                of Embryogenic Calli on M20 Medium                                            Embryonic            Kanamycin # Resistant                                    Calli      # Calli   Level (mg/l)                                                                            Surviving Calli                                ______________________________________                                        Putative   43        200       43                                             15834-     40         0        40                                             Transformed                                                                   Calli                                                                         (Example 1)                                                                   Putative   23        200       23                                             LBA4404-   10         0        10                                             Transformed                                                                   Calli                                                                         (Example 2)                                                                   Untransformed                                                                            25        200        0                                             Controls   25         0        25                                             ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        GUS Assays.sup.1                                                              Tissue     No.          No.     Percent                                       Materials  Tested       Positive                                                                              Positive                                      ______________________________________                                        Friable cells                                                                            65           65      100                                           Embryonic  38           38      100                                           Calli                                                                         Somatic    41           40       98                                           Embryos                                                                       Shoots     16           16      100                                           Plants      2            2      100                                           ______________________________________                                         .sup.1 Assays performed as described in Jefferson (1987), supra.         

Example 2 Aarobacterium tumefaciens Transformation of Rose

1. Culture tissues on callus induction medium to yield calli.

Same as Example 1.

2. Pre-embryogenic callus induction medium and their maintenance.

Same as Example 1.

3. Agrobacterium tumefaciens culture and preparation.

Same as Example 1 except Agrobacterium tumefaciens strain LBA4404(Hoekema et al. (1983), supra.) containing the binary vector pJJ3931(FIG. 2) was used for transformation. pJJ3931 is same as pJJ3499 exceptthat it carries the luciferase (-LUC) gene (Ow et al. (1986), supra.)instead of GUS, under the control of 35S promoter, used as a reportergene.

4. Cocultivation on cocultivation medium.

Same as Example 1.

5. Wash.

Same as Example 1.

6. Selection medium.

Same as Example 1 except that 25 out of 33 inoculated calli producedkanamycin-resistant calli (Table 1).

7. Culture on maintenance medium to yield somatic Embryos.

Same as Example 1.

8. Culture on maturation medium to yield mature somatic Embryos.

Same as Example 1.

9. Culture on germination medium.

Same as Example 1.

10. Culture on shoot multiplication medium.

Same as Example 1.

11. Culture on shoot elongation medium.

Same as Example 1.

12. Culture on artificial soil for root regeneration.

Same as Example 1, except shoots were cultured in Jiffy Pots saturatedwith N3-4 medium. After four weeks, complete plants were transferred tosoil.

13. Transfer to soil.

Complete plants were transferred to soil and incubated in a growthchamber (16 hr/day light, 16° C. night, 24° C. day temperature) for 2weeks. Plants were covered with plastic which was gradually removed over2 weeks to harden off the plants.

14. Results and demonstration of transformation.

Transformation was confirmed by several means: 1) transformed calli wereable to continue growth on M20 K200C medium (Table 2) and 2) mosttransformed calli tested positive and non-transformed calli testednegative in a LUC assay (Table 4 and FIG. 3)

                  TABLE 4                                                         ______________________________________                                        LUC Assay.sup.1                                                               Tissue                                                                        Materials # Tested     # Positive                                                                             % Positive                                    ______________________________________                                        Friable   15           14        93                                           Calli                                                                         Embryogenic                                                                             13           13       100                                           Calli                                                                         ______________________________________                                         .sup.1 Assays performed as described in Ow (1986), supra.                

Although the foregoing invention has been described in detail forpurposes of clarity of understanding, it will be obvious that certainmodifications may be practiced within the scope of the appended claims.

What is claimed is:
 1. A method for genetically transforming calluscells from a rose plant, said method comprising:incubating friable,granular callus cells with Agrobacterium cells carrying an exogenous DNAsequence; and selecting callus cells which express at least a portion ofthe exogenous DNA sequence.
 2. A method as in claim 1, wherein thecallus cells and the Agrobacterium cells are incubated in a mediumcontaining nutrients, an energy source, and a virulence inductioncompound for a time period from about one day to about four days.
 3. Amethod as in claim 1, wherein pre-embryogenic callus cells are incubatedwith the Agrobacterium cells.
 4. A method as in claim 1, wherein theexogenous DNA sequence includes a selectable marker gene and the calluscells are selected in a selection medium which inhibits the growth ofcells which do not express the selectable marker gene.
 5. A method forgenetically transforming a rose plant, said method comprising:(a)culturing tissue from the rose plant under conditions selected toproduce a friable, granular callus; (b) incubating cells from the callusof step (a) with Agrobacterium cells carrying an exogenous DNA sequence;(c) selecting callus cells from step (b) which express at least aportion of the DNA sequence; and (d) producing transformed plantletsfrom the selected callus cells of step (c).
 6. A method as in claim 5,wherein the tissue is derived from a plant part selected from the groupconsisting of stamen filaments, leaf explants, stem sections, shoottips, petal, sepal, petiole, and peduncle.
 7. A method as in claim 6,wherein the tissue is cultured until a hardened callus is produced,further comprising cutting the callus into sections prior to incubating.8. A method as in claim 5, wherein pre-embryogenic callus cells areincubated with the Agrobacterium cells.
 9. A method as in claim 5,wherein the exogenous DNA sequence includes a selectable marker gene andthe callus cells are selected in a selection medium which inhibits thegrowth of cells which do not express the selectable marker gene.
 10. Amethod as in claim 5, wherein the transformed plantlets are producedby:culturing the selected callus cells in a maintenance medium selectedto produce somatic embryos; culturing the somatic embryos in amaturation medium selected to produce differentiated somatic embryos;culturing the differentiated somatic embryos in a germination mediumselected to induce shoot and leaf formation on the embryos; and rootingthe germinated embryos to produce the plantlets.
 11. A rose callus cellderived from an explant material which has been transformed with anexogenous DNA sequence according to the method claim 1, said DNAsequence comprising a functional gene capable of imparting a phenotypenot possessed by the explant material, wherein said DNA sequence hasbeen integrated into the rose chromosomes.
 12. A rose plant having cellsderived from an explant material which have been transformed with anexogenous DNA sequence according to the method claim 5, said DNAsequence comprising a functional gene capable of imparting a phenotypenot possessed by the explant material, wherein said DNA sequence hasbeen integrated into the rose chromosomes.
 13. A rose callus cellproduced by the method in claim
 1. 14. A rose plant produced by themethod of claim
 5. 15. A somatic rose embryo produced by the methodcomprising:(a) culturing tissue from a rose plant on a callus inductionmedium containing nutrients, an energy source, an auxin, and a cytokininin amounts effective to induce callus formation; (b) combining cellsfrom the callus of step (a) with Aqrobacterium cells carrying theexogenous DNA sequence in a cocultivation medium containing nutrients,an energy source, and an induction compound under conditions which allowthe Agrobacterium cells to infect the callus cells and transfer theexogenous DNA sequence to the callus cell chromosomes; (c) culturingcallus cells from step (b) in a selection medium containing nutrients,an energy source, an auxin, a cytokinin, and an agent which inhibits thegrowth of callus cells which do not express the selectable marker gene;and (d) culturing the cells selected in step (c) in a maintenance mediumcontaining nutrients, an energy source, an antibacterial agent, and agrowth regulator, other than an auxin or a cytokinin, present in amountseffective to produce somatic embryos.